Abstract: :
Purpose: Matrix metalloproteinases (MMPs) are key regulators of tissueremodeling. Expression of the MMP gelatinase B (gelB, MMP–9)is induced at the migrating epithelial front and blocking itsactivity by "knock–out" of the MMP–9 gene resultsin faster epithelial resurfacing (Mohan et al., JBC, 2002).In contrast, broad–spectrum, synthetic MMP inhibitorsretard the rate of epithelial resurfacing, suggesting role forother MMPs. The purpose of this study was to identify candidateMMPs, as well as members of the TIMP family of MMP inhibitors,involved in epithelial regeneration in the mouse cornea by globalgene expression analysis.Methods: Corneal debridement was performed by demarcating a 1mm circularregion of the cornea with a trephine and removing the cornealepithelium in that region with an algebrush. The corneal stromalintegrity was visualized and ensured by topically applying 2%Fluorescein Disodium. The migrating corneal epithelium was harvested24 hours later by scraping with a scalpel dipped in TRIzol reagent(Invitrogen), and frozen at –80°C. The unwounded epitheliumfrom the contralateral eye was also harvested for comparison.Total RNA was purified from both sets of tissues the next day.Quantitative RT–PCR was performed using SYBR green asa probe. The PCR conditions were as follows: one step of denaturationfor 2 min at 95°C followed by 50 cycles of amplificationat 95°C for 15 sec, 60°C for 1 min.Results: In our first set of experiments, seven MMPs were assayed: MMP–1aand 1b, MMP–2, MMP–3, MMP–7, MMP–8,and MMP–10. The following MMPs were up–regulatedin migrating epithelium: MMP–1a (2.9 fold), MMP3 (3.4–fold),MMP–7 (2.8–fold) and MMP–10 (2.7–fold).The mRNA for MMP–1b and MMP–8 was detected onlyin the samples from migrating epithelium. We are in the processof assaying the remaining sixteen members of the MMP family,eight members of the related ADAM family, the four TIMPs, andthe related inhibitor, RECK.Conclusions: These preliminary data identify several MMPs which may be involvedin corneal epithelial regeneration. The role of these enzymeswill be assayed using gene knock–out and knock–downmethodologies.